PKR activation via hybridization chain reaction

ABSTRACT

The present application relates to the use of hybridization chain reaction (HCR) to form double stranded RNA polymers in the presence of a target, such as a nucleic acid associated with a disease or disorder. The RNA polymers are preferably able to activate the RNA-dependent kinase PKR. Activation of PKR via RNA-HCR can be used to treat a wide variety of diseases and disorders by specifically targeting diseased cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 60/725,011, filed Oct. 7, 2005, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present application relates generally to the use of hybridizationchain reaction to create RNA polymers that are able to activate theRNA-dependent kinase PKR.

2. Description of the Related Art

Hybridization Chain Reaction (HCR) is a method for the triggeredhybridization of nucleic acid molecules, typically starting frommetastable, monomer hairpins. HCR is described, for example, in U.S.patent application Ser. No. 11/087,937, filed Mar. 22, 2005 (publishedas U.S. Publication No. 2005-0260635 on Nov. 24, 2005), and Dirks andPierce (Dirks, R. and N. Pierce, Proc. Natl. Acad. Sci. USA 101(43):15275-15278 (2004)), which are incorporated herein by reference in theirentirety. In a simple version of this process, metastable monomerhairpins undergo a chain reaction of hybridization events to form anicked helix when triggered by a nucleic acid initiator strand. Afundamental principle behind HCR is that short loops are resistant toinvasion by complementary single-stranded nucleic acids. This stabilityallows for the storage of potential energy in the form of loops;potential energy is released when a triggered conformational changeallows the single-stranded bases in the loops to hybridize with acomplementary strand. One embodiment of HCR is illustrated in FIG. 1.

HCR can be used for a wide variety of purposes, for example, to create avisual signal that identifies the presence of one or more targetanalytes in a sample using nano-gold particles, as described by U.S.patent application Ser. No. 11/371,347, filed on Mar. 7, 2006, hereinincorporated by reference. It can also be used for in situ imaging anddetection applications, as described by U.S. patent application Ser. No.11/371,346, filed on Mar. 7, 2006, herein incorporated by reference. Asdescribed in detail below, it has now been found that HCR can be used tocreate RNA polymers in target cells, thereby activating the proteinkinase PKR.

Protein kinases and other cell-signaling messengers are activated duringan immune response. In vitro studies show that PKR is activated when twoPKR molecules dimerize and phosphorylate each other in complex with RNAduplexes longer than approximately 30 bp, and that the strength of theactivation increases with duplex length up to ˜85 bp (Manche, L. et al.Molecular and Cellular Biology 12:5238-5248 (1992); Wu, S., and R. J.Kaufman. The Journal of Biological Chemistry 272:1291-1296 (1997); whichare incorporated herein by reference in their entirety). Activation ofPKR can lead to selective cell death, which is a strategy envisioned forthe development of therapies for cancer (Shir, A. and A. Levitski.Nature Biotechnology 20:895-900 (2002); Friedrich, I. et al. Serminarsin Cancer Biology 14:223-230 (2004); Friedrich, I. et al. MolecularTherapy 12:969-975 (2005); which are incorporated herein by reference intheir entirety). Exploiting these activation properties, it wasdemonstrated that binding of anti-sense RNA to a region that spans thesplice point of an oncogenic mRNA fusion entity resulted in creation ofa 39-base pair duplex within cancer cells, selectively killing them(Shir, A., and A. Levitzski. 2002). The 39-bp duplex formed by theoncogenic fusion entity and the anti-sense RNA partially activated theprotein kinase PKR, which is involved in inhibition of protein synthesisand cell death. In healthy cells, binding between the anti-sense RNA andthe non-fused wild-type mRNA yielded only a 20-bp or 19-bp duplex, whichresulted in minimal PKR activation. In this approach, the duplex formedin the process of oncogene detection served as the binding site for theactivation of PKR. As a result, in this approach, the length of theactivating RNA duplex is limited to twice the length of the longestduplex that does not activate PKR; this in turn limits the extent of PKRactivation and the efficacy of a corresponding therapy.

A new approach to selective PKR activation has been developed based onthe mechanism of HCR. Metastable RNA hairpins interact upon exposure toa target molecule to form long nicked polymers that subsequentlyactivate PKR. The target molecule is preferably a mutant mRNA moleculeassociated with cancer. Activation of PKR leads to the inhibition ofprotein synthesis and to cell death (Jagus, R. et al. The InternationalJournal of Biochemistry 31:123-138 (1999); Williams, B. R. G. Oncogene18:6112-6120 (1999); which are incorporated herein by reference in theirentirety). By transducing a detection binding event into the formationof a distinct activation domain, HCR hairpins can be used that bindminimal nucleic acid sequences but generate longer activation duplexeswith multiple PKR binding domains (FIG. 5), leading to greaterspecificity and increased PKR activity. As a result, PKR is moreeffectively activated in target cells since the length of the activationduplexes is not limited as it is in the prior art approach describedabove. HCR is thus envisioned as a superior approach for activation ofPKR for the purpose of triggering the death of diseased cells.

SUMMARY OF THE INVENTION

The invention relates generally to methods, compositions and kits toinitiate hybridization chain reaction using RNA hairpin monomers tocreate RNA polymers that are able to activate the protein kinase PKR.The methods, compositions and kits can be used with samples and cellpopulations containing a target of interest. For example, RNA polymerscan be created using RNA hairpin monomers that polymerize in thepresence of a mRNA molecule associated with a disease or disorder, suchas cancer.

In some embodiments, methods of activating the RNA-dependent proteinkinase PKR are provided. A sample comprising PKR is contacted with afirst RNA hairpin monomer comprising an initiator complement region anda second RNA hairpin monomer comprising a propagation region that issubstantially complementary to a portion of the first monomer. Themonomers are preferably metastable, such that a double-stranded RNApolymer comprising the first and second monomers is only formed uponbinding of an initiator to the initiator complement region of the firstmonomer. The dsRNA polymer is preferably greater than 35 bp in lengthand thus is able to activate PKR in the sample.

The initiator is preferably able to bind to the initiator complementregion of the first monomer and stimulate polymerization when a targetis present in the sample. The target is preferably associated with adisease or disorder, and may be, for example, a nucleic acid such as anmRNA. In some embodiments the initiator is a portion of a target nucleicacid. In other embodiments the initiator is part of an initiationtrigger such as a nucleic acid probe. Upon binding of a nucleic acidprobe to a target nucleic acid sequence the initiator is made availableto bind to the first monomer and stimulate polymerization of themonomers. In other embodiments the initiator comprises a recognitionmolecule such that upon binding of the recognition molecule to thetarget the initiator is made available to bind to the first monomer andstimulate polymerization. The recognition molecule may be, for example,an aptamer. In such embodiments, appropriate targets can include, butare not limited to, proteins, carbohydrates, lipids and small molecules,as well as nucleic acids.

In some embodiments, where the target is a nucleic acid sequence, themethod of activating PKR can further include contacting the sample withat least one accessory molecule including a DNA molecule that binds toregions flanking the initiator portion of a target nucleic acidsequence.

In another aspect of the invention, methods are provided for treating adisease or disorder. Stimulation of PKR leads to reduction oftranslation and cell death. Thus, diseased cells can be targeted bystimulating PKR specifically in cells containing targets associated witha disease or disorder. In some embodiments, a disease or disorder istreated by providing a first RNA hairpin monomer to a cell comprising atarget nucleic acid associated with the disease or disorder. A secondRNA hairpin monomer is provided to the cell, wherein the second monomercomprises a propagation region that is substantially complementary to aportion of the first monomer. The first and second monomers polymerizein the presence of the target nucleic acid to form an RNA polymer thatin turn activates PKR, leading to reduced translation, reduced cellgrowth, and cell death. The methods can be carried out in vivo or invitro. The monomers may be provided to a cell, for example, inliposomes. The target nucleic acid sequence may be, for example, an mRNAassociated with a cancer or a nucleic acid associated with HIV.

In yet another aspect of the invention, kits are provided for activatingPKR in a sample comprising a target nucleic acid and PKR. The targetnucleic acid may be, for example, associated with a disease or disordersuch as cancer or HIV infection. The kits preferably comprise a firstRNA hairpin monomer comprising an initiator complement region and asecond RNA hairpin monomer comprising a propagation region that iscomplementary to a portion of the first monomer. In some embodiments theinitiator complement region is complementary to an initiator portion ofthe target nucleic acid. The RNA hairpin monomers preferably have a stemthat is from about 10 to 35 base pairs in length and a loop of at leastabout 3 base pairs. The kits may comprise additional components, such asan accessory DNA molecule that is complementary to a portion of thetarget nucleic acid molecule that flanks the initiator portion. The kitsmay also comprise a carrier that facilitates introduction of themonomers into a cell. In some embodiments an initiation trigger is alsoprovided, wherein an initiator portion of the trigger is complementaryto the initiator complement region of the first monomer and is madeavailable upon biding of the trigger to the target. The initiationtrigger may also comprise a recognition molecule, such as an aptamer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically illustrates one embodiment of an HCR system. Eachletter represents a segment of nucleic acids. Letters marked with a *are complementary to the corresponding unmarked letter.

FIG. 1A shows two hairpins, labeled H1 and H2, that are metastable inthe absence of initiator I. The hairpins comprise sticky ends ‘a’ and‘c*’, respectively. Potential energy is stored in the hairpin loops.

FIG. 1B shows how a single initiator strand ‘I’ can nucleate or bind tothe sticky end of H1 and displace one arm to open the hairpin. Thisfrees up the bases that were trapped in the hairpin, allowing them toperform a similar displacement reaction on H2.

As illustrated in FIG. 1C, the newly exposed c region of H1 nucleates atthe sticky end of H2 and opens the hairpin to expose a region on H2 (a*)that is identical in sequence to the initiator I. As a result, each copyof I can propagate a chain reaction of hybridization events betweenalternating H1 and H2 hairpins to form a nicked double helix, therebyamplifying the signal of initiator binding. The process can continueuntil the monomers (H1 and H2) are exhausted. At each step, energy isgained from the hybridization of ‘a’ or ‘c’.

FIG. 2 shows a DNA probe molecule that contains an initiator sequence(heavy dashed line) that initiates RNA-HCR upon binding of the probe toan mRNA target. The single stranded regions on either side of the duplexregion of the probe compete with native base pairing in the target mRNAto facilitate triggering of RNA-HCR.

FIG. 3 illustrates an aptamer HCR initiation trigger mechanism for thedetection of a target molecule, such as a peptide. Binding of theaptamer to the target molecule induces a conformational change thatexposes a sticky end initiator that can initiate HCR.

FIG. 4 shows how HCR-RNA monomers can be designed to polymerize by HCRupon exposure to a target. FIG. 4(a) illustrates HCR-RNA hairpinmonomers. FIG. 4(b) demonstrates delivery of the RNA hairpin monomers(‘H1’ and ‘H2’) to cells containing a target mRNA sequence (‘I’) and thelong double-stranded RNA (dsRNA) polymers that are consequently formed.These polymers can bind to and activate PKR, a human RNA-activatedprotein kinase. Once bound to dsRNA, dimerized PKR molecules activateeach other via phosphorylation and then set in motion a response thatinhibits translation and leads to cell death. FIG. 4(c) shows that incells lacking a target sequence, the HCR hairpins will not activate PKRand hence will not shut down protein synthesis or cause cell death.

FIG. 5 shows how HCR using RNA can provide a sensitive approach foractivation of PKR. An RNA hairpin monomer (‘H1’) is designed thatdetects the fusion entity (‘I1/2’) within a diseased cell. Bindingbetween H1 and I1/2 displaces one arm of H1 to open the hairpin. Thebases trapped in the hairpin are now free to bind to a complementaryregion in a second RNA hairpin monomer (‘H2’) and perform a similardisplacement reaction as described in FIG. 1. The formation of the HCRproduct by polymerization between H1 and H2 is effective at activatingPKR. Given a sufficient ratio of monomers to target molecules, polymersthat provide binding sites for multiple PKR molecules can be formed.

FIG. 6 illustrates the results of RNA-HCR polymer formation studies for(a) Ewing's sarcoma and (b) glioma.

FIG. 7 shows the results of PKR activation studies by RNA-HCR for (a)Ewing's sarcoma and (b) glioma.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Hybridization Chain Reaction (HCR) is a method for the triggeredhybridization of nucleic acid molecules starting from metastable monomerhairpins or other metastable nucleic acid structures. See, for example,Dirks, R. and N. Pierce, Proc. Natl. Acad. Sci. USA 101(43): 15275-15278(2004), and U.S. patent application Ser. No. 11/087,937, filed Mar. 22,2005 (published as U.S. Publication No. 2005-0260635 on Nov. 24, 2005),each of which is incorporated herein by reference in its entirety. HCRdoes not require any enzymes and can operate isothermally.

In one embodiment of HCR, two or more metastable monomer hairpins areused. The hairpins preferably comprise loops that are protected by longstems. The loops are thus resistant to invasion by complementarysingle-stranded nucleic acids. This stability allows for the storage ofpotential energy in the loops. Potential energy is released when atriggered conformational change allows the single-stranded bases in theloops to hybridize with a complementary strand, preferably in a secondhairpin monomer.

Each monomer is caught in a kinetic trap, preventing the system fromrapidly equilibrating. That is, pairs of monomers are unable tohybridize with each other in the absence of an initiator. Introductionof an initiator causes the monomers to undergo a chain reaction ofhybridization events to form a nicked helix (see FIGS. 1A-C).

Methods and compositions using RNA-based HCR for forming double-stranded(ds) RNA polymers and for stimulation of PKR activity by the dsRNApolymers are provided. HCR is well-suited for this type of applicationsince it can be used for the specific detection and/or treatment oftargets associated with a disease or disorder. The target, which isfound preferably exclusively in diseased cells or to a greater extent indiseased cells than in healthy cells, acts as a trigger for HCR of twoor more RNA monomers. The polymers containing dsRNA can be detected, forexample, by any of the methods described in U.S. patent application Ser.No. 11/371,347 or in U.S. patent application Ser. No. 11/371,346, eachof the foregoing which is herein incorporated by reference in itsentirety. Such detection could be used in diagnosis, for example, toconfirm the presence of the disease or disorder in a patient. Polymerformation in the presence of a target associated with a disease ordisorder can also be used therapeutically. In some embodiments, theformation of HCR dsRNA polymers in targeted cells activates PKR, leadingto inhibition of translation, reduction of cell growth and cell death.In this way, a disease or disorder can be treated and/or prevented bytargeting diseased cells.

Diseases contemplated for treatment in embodiments of the inventioninclude any disease in which a target molecule associated with thedisease is present in a cell and can initiate polymerization of HCRhairpin monomers, and wherein inhibition of translation and/or celldeath would be beneficial to a patient. The target can itself stimulateHCR polymerization or act on an initiation trigger to initiate HCR.Preferred embodiments include, but are not limited to, diseases in whichthe target is a nucleic acid molecule. In some embodiments, the nucleicacid molecule is an mRNA molecule associated with a disease or disorder,such as a mutant mRNA molecule. However, disease-associated HCR targetscan be, for example and without limitation, nucleic acid sequences,proteins, peptides, lipids, carbohydrates and small molecules.

In some embodiments, the disease to be treated is a type of cancer, suchas, for example, leukemia, carcinoma, lymphoma, astrocytoma, sarcoma andparticularly Ewing's sarcoma, glioma, retinoblastoma, melanoma, Wilm'stumor, bladder cancer, breast cancer, colon cancer, hepatocellularcancer, pancreatic cancer, prostate cancer, lung cancer, liver cancer,stomach cancer, cervical cancer, testicular cancer, renal cell cancer,and brain cancer.

In other embodiments, the disease to be treated is associated withinfection by an intracellular parasite. For example, the intracellularparasite may be a virus such as, for example, an adenovirus,cytomegalovirus, Epstein-Barr virus, herpes simplex virus, humanherpesvirus 6, varicella-zoster virus, hepatitis viruses, papillomavirus, parvovirus, polyomavirus, measles virus, rubella virus, humanimmunodeficiency virus (HIV), or human T cell leukemia virus. In otherembodiments, the intracellular parasite may be a bacterium, protozoan,fungus, or a prion. More particularly, the intracellular parasite canbe, for example, Chlamydia, Listeria, Salmonella, Legionella, Brucella,Coxiella, Rickettsia, Mycobacterium, Leishmania, Trypanasoma,Toxoplasma, and Plasmodium.

In some embodiments, HCR-RNA monomers polymerize in the presence oftarget molecules such as cancer related mRNA sequences or viral nucleicacids and form polymers that activate PKR. Activation of PKR leads togeneral inhibition of translation and cell death in cells comprising thedisease-associated target (diseased cells).

In some embodiments, the target molecule is a sequence that is necessaryfor the life cycle or replication of a virus, such as, for example, geneexpression of the virus and the expression of a cellular receptor orco-receptor necessary for viral replication. In some particularembodiment of the invention, the virus is the human immunodeficiencyvirus (HIV). The target sequence may be, for example, selected from thegroup consisting of Rev, Gag, Pol, LTRs, TAR, RRE, Ψ, att, pbs, ppt andother essential DNA and RNA cis-regulatory elements. In one embodimentof the invention, the target molecule is an expressed region of the HIVviral genome, for example, a portion of the 9-kb transcript of theintegrated HIV virus, or any of the variously spliced mRNA transcriptsof HIV (Schwartz, S; Felber, B K; Benko, D M; Fenya, E M; Pavlakis, G N.Cloning and functional analysis of multiply spliced mRNA species ofhuman immunodeficiency virus type 1. J. Virol. 1990; 64(6): 2519-29).Target regions within the HIV transcripts can be chosen to correspond toany of the viral genes, including, for example, HIV-1 LTR, vif; nef andrev.

The invention also includes methods of treating a patient suffering froma disease or disorder such as a cancer or a viral infection. In someembodiments the methods comprise administering to target cells in thepatient, such as tumor cells, an effective amount of HCR monomers, oneof the monomers having an initiator complement region having at least90% homology and preferably identical to a target region of a nucleicthat is associated with the disease, such as an mRNA associated with thecancer or a viral-associated nucleic acid. For example, the HCR RNAmonomers may be designed to polymerize in the presence of an oncogenicmRNA or a viral gene transcript.

In one embodiment, the patient to be treated is infected with the humanimmunodeficiency virus. A target cell is removed from a patient. In apreferred embodiment, the target cell is a CD34-positive hematopoieticstem cell. Such stem cells can be purified by one of skill in the art.Methods for such purification are known and taught for example in U.S.Pat. Nos. 4,965,204; 4,714,680; 5,061,620; 5,643,741; 5,677,136;5,716,827; 5,750,397 and 5,759,793. HCR RNA monomers that are able topolymerize in the presence of a target in the viral genome aretransfected into the isolated CD34-positive stem cells. The treated stemcells are then reintroduced into the patient.

Definitions

“Nucleic Acids” as used herein means oligomers of DNA or RNA. Nucleicacids may also include analogs of DNA or RNA having modifications toeither the bases or the backbone. For example, nucleic acid, as usedherein, includes the use of peptide nucleic acids (PNA). The term“nucleic acids” also includes chimeric molecules.

The term “sticky end” refers to a nucleic acid sequence that isavailable to hybridize with a complementary nucleic acid sequence. Thesecondary structure of the “sticky end” is such that the sticky end isavailable to hybridize with a complementary nucleic acid under theappropriate reaction conditions without undergoing a conformationalchange. Typically the sticky end is a single stranded nucleic acid.

“Monomers” are individual nucleic acid oligomers. Typically, at leasttwo monomers are used in hybridization chain reactions, although three,four, five, six or more monomers may be used. In some embodiments morethan two monomers are utilized, such as in HCR systems displayingquadratic and exponential growth. Typically each monomer comprises atleast one region that is complementary to at least one other monomerbeing used for the HCR reaction. The makeup of the monomers is describedin more detail below.

A first monomer in a monomer pair typically comprises an initiatorcomplement region that is complementary to a portion of an initiatormolecule. The initiator complement region is preferably a sticky end.Other embodiments of the first monomer comprise a recognition moleculethat binds or interacts with an initiator molecule. Interaction of theinitiator to the initiator complement region or to the recognitionmolecule begins an HCR reaction.

In addition, the second monomer in the pair preferably comprises apropagation region that is able to hybridize to a complementary regionof another monomer, preferably another copy of the first monomer, tocontinue the HCR reaction begun by the initiator. The propagation regioncan be, for example, the loop region of a hairpin monomer as describedbelow. In one embodiment the propagation region on the second monomer isidentical to the portion of a nucleic acid initiator molecule that iscomplementary to the initiator complement region of the first monomer.

The propagation region on the second monomer is preferably onlyavailable to interact with the initiator complement region of the firstmonomer when an HCR reaction has been started by an interaction betweenthe initiator and the first monomer. That is, the propagation regionbecomes available to hybridize to a complementary region of anothermonomer, preferably another copy of the first monomer, when one copy ofthe first monomer has already hybridized to a second monomer, asdiscussed in more detail below.

Preferred monomers are “metastable.” That is, in the absence of aninitiator they are kinetically disfavored from associating with othermonomers comprising complementary regions. “HCR” monomers are monomersthat are able to assemble upon exposure to an initiator to form apolymer.

As used herein, “polymerization” refers to the association of two ormore monomers to form a polymer. The “polymer” may comprise covalentbonds, non-covalent bonds or both. For example, in some embodiments twospecies of monomers are able to hybridize in an alternating pattern toform a polymer comprising a nicked double helix. The polymers are alsoreferred to herein as “HCR products.”

An “initiator” is a molecule that is able to initiate the polymerizationof monomers. Initiators include molecules that comprise a region thathybridizes, binds or otherwise interacts with the initiator complementregion or recognition molecule of an HCR monomer. In some embodiments,the initiator is a combination of molecules that can initiate HCRpolymerization. In other embodiments, the initiator is a portion of aninitiation trigger, where interaction between the trigger and a targetexposes the initiator to stimulate HCR polymerization. Preferredinitiators comprise a nucleic acid region that is complementary to theinitiator complement region of an HCR monomer.

A “target” is a molecule of interest, or a combination of molecules ofinterest, whose presence can trigger HCR polymerization. Preferredtargets are associated with a disease or disorder. In some embodiments,the target comprises an initiator that is able to stimulate HCRpolymerization of the monomers. In other embodiments, the target is amolecule, or combination of molecules, that is recognized by theinitiator, or an initiation trigger comprising the initiator, such thatthe initiator is made available to induce HCR polymerization.

A “target cell” is a cell that contains a target associated with adisease or disorder. The target is typically one that is not normallyfound in healthy cells or that is found to a greater extent in diseasedcells than in healthy cells. The target can act as the initiator for HCRwhen HCR monomers are introduced into the target cell. Examples oftarget cells include cells that contain a nucleic acid signature for adisease, such as, for example, mutant mRNA or fusion mRNA entities.Other examples include, but are not limited to, cells that containhigher-than-background levels of peptides, polypeptides, antibodies orfragments thereof, signal cascade molecules, viral particles, bacteriaand parasitic organisms.

Monomers

Two or more distinct species of nucleic acid monomers are preferablyutilized in an HCR reaction. In the methods described herein, themonomers are preferably RNA monomers. Each monomer species typicallycomprises at least one region that is complementary to a portion ofanother monomer species. However, the monomers are designed such thatthey are kinetically trapped and the system is unable to equilibrate inthe absence of an initiator molecule that can disrupt the secondarystructure of one of the monomers. Thus, the monomers are unable topolymerize in the absence of the initiator. Binding of an initiatorspecies to a monomer triggers a chain reaction of alternating kineticescapes by the two or more monomer species resulting in formation of apolymer. In the examples below, two RNA hairpin monomers polymerize inthe presence of an initiator to form a nicked, double-stranded RNAhelix.

In a preferred embodiment, two or more monomer species are employed thathave a hairpin structure. The hairpin monomers preferably comprise loopsprotected by long stems. In other embodiments, monomers with a differentsecondary structure are provided. However, the secondary structure ispreferably such that the monomers are metastable under the reactionconditions in the absence of an initiator. In the presence of aninitiator, the secondary structure of a first monomer changes such thatit is able to hybridize to a sticky end of a second monomer species.This in turn leads to a change in the secondary structure of the secondmonomer, which is then able to hybridize to another monomer and continuethe process. In preferred embodiments, the second monomer is able tohybridize to another copy of the first monomer to continue thepolymerization process. In this way, once a single copy of the firstmonomer interacts with a single copy of the initiator, a chain reactionis produced such that the monomers are able to assemble into a polymercomprising alternating monomer species. Thus, the presence of multipletarget molecules, multiple polymers are produced.

A number of criteria can be used to design the monomers to achieve thedesired properties. These include, for example and without limitation,sequence symmetry minimization, the probability of adopting the targetsecondary structure at equilibrium, the average number of incorrectnucleotides at equilibrium relative to the target structure, andhybridization kinetics.

Monomers can be synthesized using standard methods, includingcommercially available nucleic acid synthesizers or obtained fromcommercial sources such as Integrated DNA Technologies (Coralville,Iowa).

In preferred embodiments, the monomers are RNA monomers. In someembodiments, the monomers contain a fluorophore, luminescent molecule,calorimetric compound or other component that allows the resultingpolymers to be visualized.

In preferred embodiments, at least two RNA hairpin monomers are utilizedas illustrated in FIG. 1A. The monomers each preferably comprise asticky end (a and c*, respectively), a first complementary segment (band b*, respectively), a loop segment (c and a*, respectively), and asecond complementary segment (b and b*, respectively). The first andsecond complementary segments are also referred to as “stems” andtogether form a duplex region.

The first monomer (H1) preferably comprises a sticky end a that iscomplementary to a first nucleic acid portion a* of an initiator (I;FIG. 1B). This sticky end is referred to herein as the “initiatorcomplement region.” The initiator may be, for example, a target ofinterest, a nucleic acid that is able to contact the first monomer onlyin the presence of a target of interest, or any cellular component (suchas, for example, a nucleic acid sequence) that is found only in targetdiseased cells or to a lesser extent in healthy cells, as discussed inmore detail below.

The second monomer (H2) preferably comprises a sticky end c* that iscomplementary to a portion of the first monomer that becomes accessibleupon initiator binding. Preferably the sticky end c* is complementary tothe loop segment c of the first monomer (FIG. 1A). The loop segment c ofthe first monomer is preferably not available to hybridize with stickyend c* of the second monomer in the absence of initiator.

The first and second complementary segments (b and b*) in the first andsecond monomers are typically substantially identical. That is, thefirst complementary segment b of the first monomer (H1) is able tohybridize to the second complementary segment b* of the second monomer(H2).

The first complementary segment of each monomer is also able tohybridize to the second complementary segment of the same monomer toform the hairpin structure. For example, as shown in FIG. 1A, the firstmonomer (H1) comprises a first complementary segment b that is able tohybridize to the second complementary segment b*. In the absence of aninitiator, the first and second complementary segments of each monomerare generally hybridized to form a duplex region of the metastablemonomer.

In some embodiments, the first complementary segment b of the firstmonomer is also complementary to a portion b* of the initiator, suchthat upon hybridization of the initiator region a* to the sticky end a(the initiator complement region) of the first monomer H1, one arm ofthe hairpin structure is displaced. This opens the hairpin and allowsbinding of the first complementary segent b to the second portion b* ofthe initiator strand (FIG. 1B). In other embodiments, a recognitionmolecule included within the first monomer can hybridize, bind orinteract with at least a portion of the initiator such that one stem ofthe hairpin structure is displaced, allowing opening of the hairpin.

Recognition molecules include, without limitation, polypeptides, such asantibodies and antibody fragments, nucleic acids, aptamers, and smallmolecules. The use of a first HCR monomer bound to a recognitionmolecule is described in more detail below.

The loop segment c of the first monomer is also exposed by the openingof the hairpin and is able to bind to the sticky end c* of the secondmonomer H2, as illustrated in FIG. 1C. This opens the second monomerhairpin H2 and the second complementary segment b* of the first monomeris able to hybridize to the first complementary segment b of the secondmonomer H2.

This leaves the loop region a* and first complementary region b* of thesecond monomer H2 exposed (FIG. 1C). The sticky end a of another firstmonomer (H1) species is able to bind to the exposed loop region a* ofthe second monomer H2, thus opening the H1 hairpin and continuing theprocess described above. Because the loop region a of the second monomeracts as an initiator on a second H1 monomer and allows the process tocontinue in the absence of further initiator, it is referred to as thepropagation region.

At each step, energy is gained from the hybridization of the sticky endof the monomer. The result is a nicked, double helix RNA polymercomprising alternating H1 and H2 fragments. This process preferablycontinues in a chain reaction until all of one or both of the monomerspecies is used up, or the reaction is stopped by some other mechanism.If desired, the nicks in the nucleic acid polymer structures that resultfrom HCR can by ligated (for example, using ligase).

Because of the self-propagating nature of the reaction, each copy of theinitiator species can begin the chain reaction. Further, as long asthere is a fixed supply of monomers the average length of the resultingpolymers is inversely related to the initiator concentration. Theconcentration of the monomers can be adjusted to ensure that thepolymers are of sufficient length to activate one or more PKR molecules,more preferably two or more PKR molecules, for each target that isdetected.

The length of the loop, stem and sticky ends of the monomers can beadjusted, for example to ensure kinetic stability in particular reactionconditions and to adjust the rate of polymerization in the presence ofinitiator. In one preferred embodiment the length of the sticky ends isthe same as the length of the loops. In other embodiments the stickyends are longer or shorter than the loops. However, if the loops arelonger than the sticky ends, the loops preferably comprise a region thatis complementary to the sticky end of a monomer.

In some preferred embodiments the length of the loops is short relativeto the stems. For example, the stems may be two or three times as longas the loops.

The loop regions are preferably between about 1 and about 100nucleoteides, more preferably between about 3 and about 30 nucleotidesand even more preferably between about 4 and about 7 nucleotides. In oneembodiment the loops and sticky ends of a pair of hairpin monomers areabout 6 nucleotides in length and the stems are about 18 nucleotideslong.

Other refinements to the system stabilize the monomer hairpins to helpprevent HCR in the absence of an initiator. This can be achieved, forexample, via super-stable hairpin loop sequences (Nakano et al.Biochemistry 41:14281-14292 (2002), herein incorporated by reference inits entirety), with ostensible structural features that could furtherinhibit direct hybridization to the hairpin. In other embodimentshairpin loops are made to be self-complementary at their ends. Thisself-complementation “pinches” the hairpin loops, making them shorter.However, if the reactive sticky ends of each monomer are complementaryto the loop regions on the opposite monomer, as described above, theywill have a slight propensity to close up, thereby slowing down thereaction. This feature can be utilized if a slower reaction is desired.Completely self-complementary hairpins can also be used, for example ifthe monomer hairpins are forming dimers with interior loops that aremore easily invaded than their hairpin counterparts.

Reaction conditions are preferably selected such that hybridization isable to occur, both between the initiator and the sticky end of a firstmonomer, and between the complementary regions of the monomersthemselves. The reaction temperature does not need to be changed tofacilitate the hybridization chain reaction. That is, the HCR reactionsare isothermic. They also do not require the presence of any enzymes.

Variations

There are many possible variations to HCR that may improve its speed,stability and ability to activate PKR. The system illustrated in FIG. 1and discussed above exhibits linear growth in response to initiator.However, increasing the rate of polymer growth can enhance the abilityto respond to the presence of low copy number targets, such as a singletarget molecule in a large volume. For example, monomers can be designedto undergo triggered self-assembly into branched structures exhibitingquadratic growth or dendritic structures exhibiting exponential growth.The exponential growth is limited by the available space such that itdecreases to cubic amplification as the volume around the initiatorfills. However, if chain reactions products are able to dissociate,exponential growth can be maintained until the supply of monomers isexhausted.

In order to achieve non-linear growth, 3 or more HCR monomers can beused. In preferred embodiments, at least 4 HCR monomers are used. Insome embodiments, at least one monomer in a primary monomer pairincorporate a nucleic acid trigger segment that is complementary to theexposed sticky end of one of the monomers from a secondary set of HCRmonomers. Upon exposure to the nucleic acid that is to be detected, theset of primary monomers undergoes HCR to form a polymer with a periodicsingle stranded trigger region. Thus the trigger nucleic acid isexposed, leading to a polymerization chain reaction in the secondary setof monomers. In other embodiments, both the primary and secondary set ofmonomers includes a trigger segment, such that exponential growth isachieved. Exemplary schemes are presented and described in U.S. patentapplication Ser. No. 11/087,937 (published as U.S. Publication No.2005-0260635), herein incorporated by reference in its entirety.

Initiator

The initiator is preferably a nucleic acid or other molecule that isable to contact the first monomer and trigger polymerization only in thepresence of a target associated with a disease or disorder. The targetcan be, but is not limited to, any of the following: a nucleic acidsequence, a peptide, a polypeptide, an antibody or fragment thereof, asignal cascade molecule, a lipid, a carbohydrate, a fused entity, aviral particle, a bacterium or a parasitic organism. In preferredembodiments, the initiator is at least a portion of the target. Forexample, in one embodiment, the initiator can be a portion of a nucleicacid associated with a disease or disorder.

The initiator is preferably a nucleic acid molecule. The nucleic acidinitiator comprises an initiator region that is complementary to aportion of an HCR monomer, preferably a portion of the monomer that isavailable for hybridization with the initiator while the monomer is inits kinetically stable state. The initiator also preferably comprises asequence that is complementary to a portion of the monomer adjacent tothe sticky end such that hybridization of the initiator to the stickyend causes a conformational change in the monomer and begins the HCRchain reaction. For example, the initiator may comprise a region that iscomplementary to the first complementary region of the HCR monomer, asdescribed above and illustrated in FIG. 1.

In the preferred embodiments, the sequence of the initiator iscomplementary to the sticky end (initiator complementary region) andfirst complementary region of a first monomer. As described above, insome embodiments this will also influence the sequence of the secondcomplementary region and the loop of the second monomer species.

In some embodiments the initiator is a target nucleic acid or a portionof a target nucleic acid that is to be detected in a sample (FIG. 4). Inthis case, the sequence of the target nucleic acid is taken intoconsideration in designing the HCR monomers. For example, the initiatorcomplement region, preferably a sticky end, of one monomer (‘H1’) isdesigned to be complementary to a portion of the target nucleic acidsequence (‘I’). The initiator complement region is preferably at least80%, more preferably at least 90%, 95% or higher, identical to theinitiator sequence. In preferred embodiments, the initiator sequence isat least 2, 3, 4, 5, or 10 or more bases in length.

Similarly, a region adjacent to the sticky end of the same monomer canbe designed to be complementary to a second region of the targetsequence as well. Because the second monomer (‘H2’) will hybridize tothe first monomer (‘H1’), the sequence of the second monomer will alsoreflect at least a portion of the sequence of the target nucleic acidand/or the initiator. In preferred embodiments, the initiator is an mRNAtarget sequence or a portion of an mRNA target sequence, for example, aportion of a mutant mRNA sequence that comprises a mutation associatedwith a disease or disorder. Embodiments also include specificcombinations of mRNA sequences.

In other embodiments, the initiator comprises at least a portion of anucleic acid that is part of an “initiation trigger” such that theinitiator is made available when a predetermined physical event occurs.In the preferred embodiments, that predetermined event is the presenceof a target associated with a disease or disorder. In each of theseembodiments, the initiator is preferably associated with a molecule thatis responsive to the presence of the target. Thus, the initiator and theassociated molecule together form the initiation trigger. For example,the initiator may be associated with a molecule that undergoes aconformational change in response to binding to the target (FIG. 2). Theconformational change exposes the initiator and thereby stimulatespolymerization of the HCR monomers. In other embodiments, however, theinitiation trigger comprises a single nucleic acid. The initiator regionof the nucleic acid is made available in the presence of the target.

The structure of the initiation trigger is preferably such that when thetarget is not present (or the other physical event has not occurred),the initiator is not available to hybridize with the sticky end of amonomer. The target frees the initiator such that it can interact with ametastable monomer, triggering the HCR polymerization reactionsdescribed above. In some embodiments, the target causes a conformationalchange in the trigger that allows the initiator to interact with themonomer.

The initiator may be part of a trigger comprising a nucleic acidinitiator that is linked to or associated with a recognition molecule,such as an aptamer, that is capable of interacting with a target ofinterest (FIG. 3). The trigger is designed such that the initiator isunavailable to stimulate HCR in the absence of the target. When thetarget interacts with the recognition molecule, the initiator is able tostimulate HCR. Preferably, the recognition molecule is one that iscapable of binding the target.

Recognition molecules include, without limitation, polypeptides,antibodies and antibody fragments, nucleic acids, aptamers, and smallmolecules. The use of an initiator bound to an aptamer is described inmore detail below.

In some particular embodiments, amplification is achieved by coupling afirst HCR monomer to a recognition molecule such as, for example, anucleic acid aptamer trigger. An aptamer is identified that is able tospecifically bind the target. In such embodiments, the target is notlimited to a nucleic acid but may be, for example, a polypeptide orsmall molecule. The aptamer is linked to the HCR monomer in such a waythat the initiator is unavailable to stimulate HCR in the absence oftarget binding to the aptamer.

Preferably, conformational changes in the first HCR monomer-aptamersecondary structure expose the first complementary segment. In oneembodiment, such an aptamer trigger is a hairpin nucleic acid thatcomprises a first complementary segment that is able to hybridize to asecond complementary segment of a second HCR monomer. The hairpinaptamer trigger may also comprise a region that enhances the stabilityof the hairpin in the absence of aptamer binding to the initiator, suchas a nucleic acid region in one arm of the hairpin that is complementaryto a region of the other arm.

FIG. 2 illustrates how a DNA probe molecule containing a triggersequence (heavy dashed line) can initiate RNA-HCR polymerization uponbinding to a target mRNA molecule. The single stranded regions on eitherside of the duplex region of the probe compete with native base pairingin the target molecule to facilitate triggering of the polymerizationreaction.

FIG. 3 depicts a scheme for HCR amplification using an aptamer constructthat exposes an initiator strand upon binding to a target. The stickyend can act as a trigger for the HCR mechanism of FIG. 1 by openinghairpin H2. The region X is introduced to help stabilize the trigger inthe absence of analyte. The region b* includes both the hairpin loop andthe portion of the stem complementary to X. This trigger mechanism isbased on conformational changes in the aptamer secondary structure (Li,Y. Journal of the American Chemical Society 125:4771-4778 (2003), hereinincorporated by reference in its entirety) that make the initiatorstrand available to stimulate HCR.

Stimulation of PKR Activity Using RNA-Based HCR Monomers

Long stretches of double stranded RNA are known to trigger an interferonresponse in mammalian cells via the RNA-dependent protein kinase PKR.This is a part of a natural anti-viral response meant to inhibittranslation when an infection is detected. This response inhibits cellgrowth, and leads to programmed cell death (apoptosis). Sufficientlyshort RNAs such as, for example, HCR hairpins with stems of approximatelength 14, do not effectively induce an interferon response.Consequently, if RNA-based HCR hairpins are delivered to a mixedpopulation of cells where some contain a specific target molecule andothers do not, then HCR polymerization and a subsequent interferonresponse will only occur in cells containing the trigger and thus whereRNA polymers are formed. In this way, inhibition of cell growth and/orcell death can be induced specifically in diseased cells, such as tumorcells, using HCR, while avoiding the the response in other cells. Thistechnique can be used as a therapeutic to inhibit or kill diseased orcancerous cells, leaving healthy cells intact as unpolymerized RNAhairpins eventually degrade harmlessly via natural mechanisms.

HCR is therefore considered an effective strategy for activating PKRwithin target cells by forming long RNA HCR assemblies within the cells.Once an HCR product forms within a target cell, the HCR product servesas an activation domain to upregulate PKR, leading to subsequentinhibition of translation and apoptosis. Design of appropriate HCRhairpin monomers that do not themselves activate PKR, but activate PKRupon binding a target can be derived from target sequences well known inthe art and available from literature reviews and disclosed in, forexample, various databases (e.g. NCBI).

HCR can be performed using RNA hairpin monomers to detect a targetassociated with a disease or a disorder. In some embodiments, the targetis associated with a membrane. In other embodiments, the target is anon-tethered entity. The target can be, for example, a nucleic acidassociated with cancer, such as an mRNA associated with a cancer. Inembodiments where the target is also the initiator, a first RNA hairpinwith an initiator complement region hybridizes or binds the target. Inembodiments where the target and initiator are distinct entities,recognition of the target by the initiator or by the trigger comprisingthe initiator allows binding between a first RNA hairpin comprising aninitiator complement region and the initiator. Binding between theinitiator complement region of the first hairpin and the initiator opensthe hairpin to expose a sequence that is substantially complementary tothe propagation region of a second RNA hairpin monomer.

Hybridization between the exposed sequence of the first hairpin monomerand the propagation region of the second hairpin monomer opens thesecond hairpin. This event exposes a sequence in the second hairpin thatis substantially complementary to the initiator complement region onanother copy of the first hairpin. Hybridization consequently occursbetween the exposed sequence of the second hairpin and the initiatorcomplement of another copy of the first hairpin, and the chain reactioncontinues as described until the supply of hairpin monomers isexhausted. The HCR product that forms is a nicked double-stranded RNApolymer. The double-stranded RNA products that form in cells containingthe target are of a length that is sufficient to activate one or morePKR molecules per polymer product. In preferred embodiments, the RNAproducts comprise at least 35 base pairs. Activation of PKR can lead toinhibition of translation, inhibition of cell growth and to cell death.

Double-stranded RNA polymers formed by HCR are at least approximately 35bp in length. In some embodiments, the RNA polymers are at leastapproximately 30 bp in length. In other embodiments, the RNA polymersare at least approximately 25 bp in length. In further embodiments, theRNA polymers are at least approximately 20 bp in length. In still otherembodiments, the RNA polymers are at least approximately 15 bp inlength.

In some embodiments, HCR is used to treat a patient suffering from adisease or disorder. Because HCR polymerization is specifically limitedto those cells containing the target associated with the disease ordisorder, the HCR monomers can be delivered to a multitude of cells,including healthy cells. Thus, general delivery of the monomers to apopulation of cells comprising cells containing a disease-associatedtarget and wild-type cells is possible. HCR polymerization takes placein diseased cells containing the target, leading to the death of thosecells in therapeutic treatments. In a particular embodiment, HCRpolymerization occurs in target cells comprising an mRNA moleculeassociated with cancer, leading to the death of cancerous cells.

Delivery of HCR Monomers to Target Cells

HCR monomers and any accessory molecules, such as, for example,initiation triggers, can be formulated with any of a variety of carrierswell known in the art to facilitate introduction into a cell. Suitablecarriers for delivery of nucleic acids to cells are well known in theart and include, for example, polymers, proteins, carbohydrates andlipids. For example, a cyclodextrin-containing polymer can be used forthe delivery of the nucleic acid HCR monomers. Commercial transfectionreagents known in the art, such as, for example, LNCaP (AltogenBiosystems) or lipofectamine RNAiMax (Invitrogen), can be used.

Delivery of nucleic acids can be accomplished, for example, as describedby Heidel (Heidel, J. D. 2005. Targeted, systematic non-viral deliveryof small interfering RNA in vivo. Doctoral thesis, California Instituteof Technology. 128 p., herein incorporated by reference in itsentirety). Also contemplated within the scope of the subject matter aregene delivery systems as described by Felgner et al. (Felgner et al.1997. Hum Gene Ther 8:511-512, herein incorporated by reference in itsentirety), including cationic lipid-based delivery systems (lipoplex),polycation-based delivery systems (polyplex) and a combination thereof(lipopolyplex). Cationic lipids are described, for example, in U.S. Pat.Nos. 4,897,355 and 5,459,127, each of the foregoing which is hereinincorporated by reference in its entirety. Proteins can also be used forHCR delivery, such as synthetic neoglycoproteins (Ferkol et al. 1993.FASEB J 7:1081-1091; Perales et al. 1994. Proc Nat Acad Sci91:4086-4090; each of which is incorporated herein by reference in itsentirety). epidermal growth factor (EGF) (Myers, EPO 0273085,incorporated herein by reference in its entirety), and other ligands forreceptor-mediated gene transfer (Wu and Wu. 1987. J Biol Chem262(10):4429-4432; Wagner et al. 1990. Proc Natl Acad Sci USA87(9):3410-3414; Ferkol et al. 1993. J. Clin Invest 92(5):2394-2300;Perales et al. 1994. Proc Natl Acad Sci USA 91(9):4086-4090; Myers, EPO0273085; each of which is incorporated herein by reference in itsentirety).

Viral and viral vector-like delivery systems generally known in the art,such as those described, for example, in U.S. Pat. No. 7,0333,834; U.S.Pat. No. 6,899,871; U.S. Pat. No. 6,555,367; U.S. Pat. No. 6,485,965;U.S. Pat. No. 5,928,913; U.S. patent application Ser. No. 10/801,648;U.S. patent application Ser. No. 10/319,074, and U.S. patent applicationSer. No. 09/839,698, each of which is herein incorporated by reference,are also contemplated for use in the present subject matter. Inaddition, standard electroporation techniques can be readily adopted todeliver HCR monomers.

Delivery of HCR monomers can occur in vivo or ex vivo. In someembodiments, cells can be removed from a patient, transfected with themonomers and returned to the patient for therapeutic effects. In otherembodiments, HCR monomers can be delivered to cells in vivo such as by,for example, injection of the monomers within a delivery vehicle intothe bloodstream or by intramuscular, subcutaneous, or intraperitonealmeans. An appropriate means of delivering HCR monomers to a desiredpopulation of cells can be identified by the skilled practitioner basedon the particular circumstances without undue experimentation.

HCR Initiators and Initiation of HCR

Target strands of mRNA can serve as initiators of HCR. Embodiments ofHCR initiators also include subsequences of mRNA. Based on thedisease-associated sequence that is to be targeted, HCR monomers can bedesigned that only polymerize in the presence of mRNA containing thetargeted sequence. If an mRNA subsequence is chosen that is onlyexpressed in certain types of cells e.g. cells with a particular geneticmutation or nucleic acid signature, then HCR will only occur in thetargeted cells. Polymerization of HCR monomers will not occur in cellswithout the target. HCR initiated by the target mRNA sequence creates alarge HCR assembly, which can activate PKR. PKR is believed to cause anatural immune response that inhibits protein synthesis and leads tocell death.

Targets contemplated for initiation of HCR are those associated with adisease or disorder, as described in detail above. In some embodiments,the target is a nucleic acid signature or specific mutation in a geneticsequence associated with a disease or disorder. Genetic mutationsinclude, but are not limited to, point mutations, non-native geneticfusions, deletion of at least one base, insertion of at least one base,frame-shift mutations, and inversions. In other embodiments, the targetis a combination of nucleic acid molecules associated with a disease ordisorder.

Targets for initiation of HCR also include, but are not limited to,nucleic acid molecules, proteins, peptides, carbohydrates, lipids andsmall molecules. In some embodiments, the target acts as the initiatorfor HCR by binding directly to the initiator complement region of an HCRmonomer to stimulate HCR polymerization. In other embodiments, thetarget binds a molecule that is associated with an initiator of HCRpolymerization.

Where the disease to be treated is a cancer, an mRNA target is typicallyone expressed in cancer cells and not in healthy cells or at least to alesser extent in healthy cells. In some cases, a disease may beidentified by the expression of several mRNA targets simultaneously. Inthis case, the HCR trigger sequence can be designed to initiate HCR onlyin the event that a specific combination of mRNAs is detected, forexample, by detecting a portion of an mRNA fusion entity.

HCR can be performed with RNA monomers instead of, or in conjunctionwith, DNA monomers. For the basic HCR scheme shown in FIG. 4, RNAhairpins with stems of approximate length 14 and loops of approximatelength 4 exhibit similar properties to DNA hairpins with stems ofapproximate length 18 and loops of approximate length 6. Stems and loopsof other lengths are also possible.

In some embodiments, RNA monomers are used in conjunction with nucleicacid “helper” monomers to facilitate target recognition (FIG. 6). Inpreferred embodiments, the nucleic acid “helper” monomers are DNAmolecules. It has been observed that HCR can be more difficult toinitiate using a long mRNA or other nucleic acid target becausesecondary structure within the mRNA strand reduces accessibility of thetarget site to the HCR monomers. The use of DNA “helper” monomers thatbind to regions flanking the target site helps eliminate competingsecondary structures that form between the target site and the flankingregions. HCR is more effectively initiated as a result of theelimination of secondary structure formation within the target mRNAstrand. Helper DNA strands can be from about 10 to about 100 bases inlength. In some embodiments, the helper DNA strands are from about 10 toabout 75 bases in length. In other embodiments, the helper DNA strandsare from about 10 to about 50 bases in length. In other embodiments, thehelper DNA strands are from about 10 to about 35 bases in length. Inpreferred embodiments, the helper DNA strands are from about 10 to about25 bases in length.

In some embodiments, initiation of HCR involving RNA monomers are usedin conjunction with a trigger comprising a DNA probe molecule thatcontains a trigger sequence (FIG. 2). The trigger sequence in the DNAmolecule, illustrated as a heavy dashed line in FIG. 2, is exposed uponbinding between the DNA molecule and a target mRNA molecule and acts asan initiator to stimulate RNA-HCR polymerization. The single strandedregions on either side of the duplex region of the DNA probe competewith native base pairing within the target molecule to facilitatetriggering of the polymerization reaction.

The design of the RNA hairpin monomers can be adjusted such that theybind specifically to nucleic acid targets, mRNA or otherwise. The designcan be derived from sequences derived from literature reviews anddisclosed in, for example, various databases (e.g. NCBI).

Compositions and Kits for PKR Activation and Therapeutic Benefit

Compositions and kits for recognition of targets associated with adisease or disorder and activation of PKR are contemplated for usewithin the scope of the subject matter. In preferred embodiments, thecompositions comprise a first RNA hairpin monomer and a second RNAhairpin monomer. Upon delivery to a target cell (or sample) andrecognition of the target, HCR is initiated causing the first and secondhairpin monomers to form long, nicked double stranded RNA polymers thatare able to bind and activate one or more PKR molecules for each targetthat is detected.

The compositions can also contain other components, such as, forexample, accessory molecules that facilitate target recognition and aidthe formation of HCR polymer products. Accessory molecules typicallycomprise nucleic acid molecules. In some embodiments, the accessorymolecules are DNA helper strands that bind to regions flanking a targetnucleic acid sequence. In other embodiments, the accessory molecule is aDNA probe molecule that contains a trigger sequence that initiates HCRupon interaction between the DNA probe molecule and a target.

Furthermore, the composition can comprise a carrier that facilitates theintroduction of nucleic acids, such as, for example, RNA hairpinmonomers and accessory nucleic acid molecules, into a cell containing atarget associated with a disease or disorder. Carriers for delivery ofnucleic acids into cells are well known in the art and described above.

A kit for recognition of a target and activation of PKR typicallycomprises the compositions as described in detail above. In preferredembodiments, the kit is used to deliver HCR hairpin monomers to apopulation of cells comprising cells comprising a disease-associatedtarget as well as healthy, wild-type cells. In some embodiments, the kitis used to deliver HCR hairpin monomers to the tissues of a patient,wherein the tissues comprise cells comprising a target associated with adisease or disorder. In other embodiments, the kit is used to select forcells containing a target in vitro.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and fall within the scope of theappended claims.

EXAMPLES Example 1 Polymer Formation Studies for Ewing's Sarcoma andGlioma

Polymer formation studies to observe HCR polymerization in the presenceand absence of target mRNA were carried out. Stock DNA/RNA solutionswere prepared as follows: hairpin 1 (“H1”), hairpin 2 (“H2”),disease-associated target mRNA (either “RNA initiator” comprising only atarget sequence without flanking regions or “target mRNA fusion”comprising the target sequence with flanking regions), mRNA 1 (“healthymRNA 1”), mRNA 2 (“healthy mRNA 2”), and helper DNA (“D1” and “D2”annealed together in one solution). Reactions were set up by combiningDNA/RNA components as indicated below. The samples were loaded on 10%TBE-PAGE gels, stained with SYBR gold and imaged on a Fuji fluorescentgel scanner for analysis.

FIG. 6 demonstrates the results of the polymer formation studies fortargets from (a) Ewing's sarcoma and (b) glioma. The results demonstratethat RNA-HCR polymerization does not proceed in the absence of thedisease-associated target sequence (Lane 3). Polymerization is observedif the target sequence is introduced without flanking regions (Lane 4)but is not observed if a longer mutant mRNA fusion is used that includesflanking regions (Lane 5) due to competing secondary structure thatforms in the mRNA. Introduction of DNA helper strands that bind to theflanking regions on either side of the target sequence restores theRNA-HCR polymerization (Lane 6). The two healthy mRNAs that do notcontain the fusion do not cause polymerization (Lanes 7 and 8). Thehelper DNAs also fail to cause poymerization (Lane 9).

The sequences of the DNA/RNA components used for the studies are asfollows: Ewing's sarcoma RNA initiator: 5′-AGCAGAACCCUUCUUAUG-3′ (SEQ IDNO: 1) RNA hairpin H1: 5′-CAGACAUAAGAAGGGUUCAGCAGAACCCUU (SEQ ID NO: 2)CUUAUG-3′ RNA hairpin H2: 5′-GAACCCUUCUUAUGUCUGCAUAAGAAGGGU (SEQ ID NO:3) UCUGCU-3′ Target mRNA fusion: 5′-CAGAGCAGCAGCUACGGGCAGCAGAACCCU (SEQID NO: 4) UCUUAUGACUCAGUCAGAAG-3′ (targeted region is underlined withflanking regions on either side) Healthy mRNA1:5′-AUAUAGCCAACAGAGCAGCAGCUACGGGCA (SEQ ID NO: 5) GCAGAGUUCAUUCCUUCCGA-3′(subsequence that is part of targeted region in the oncogenic fusion isunderlined) Healthy mRNA2: 5′-UGAGUGUCAAAGAAGACCCUUCUUAUGACU (SEQ ID NO:6) CAGUCAGAAGAGGAGCUUGG-3′ (subsequence that is part of targeted regionin the oncogenic fusion is underlined) Helper DNA D1:5′-CTTCTGACTGAGT-3′ (SEQ ID NO: 7) Helper DNA D2:5′-GCCCGTAGCTGCTGCTCTG-3′ (SEQ ID NO: 8) Glioma RNA initiator:5′-AAAAGAAAGGUAAUUAUG-3′ (SEQ ID NO: 9) RNA hairpin H1:5′-CAUAAUUACCUUUCUUUUGGGCAAAAGAAA (SEQ ID NO: 10) GGUAAU-3′ RNA hairpinH2: 5′-AAAAGAAAGGUAAUUAUGAUUACCUUUCUU (SEQ ID NO: 11) UUGCCC-3′ TargetmRNA fusion: 5′-AGUCGGGCUCUGGAGGAAAAGAAAGGUAAU (SEQ ID NO: 12)UAUGUGGUGACAGAUCACGG-3′ (targeted region is underlined with flankingregions on either side) Healthy mRNA1: 5′-CUGCCCGGCGAGUCGGGCUCUGGAGGAAAA(SEQ ID NO: 13) GAAAGUUUGCCAAGGCACGA-3′ (subsequence that is part oftargeted region in the oncogenic fusion is underlined) Healthy mRNA2:5′-UGAAGAAGUGUCCCCGUAAUUAUGUGGUGA (SEQ ID NO: 14)CAGAUCACGGCUCGUGCGUC-3′ (subsequence that is part of targeted region inthe oncogenic fusion is underlined) Helper DNA D1:5′-CCGTGATCTGTCACCA-3′ (SEQ ID NO: 15) Helper DNA D2:5′-CCTCCAGAGCCCGACT-3′ (SEQ ID NO: 16)

Example 2 PKR Activation Studies for Ewing's Sarcoma and Glioma

PKR activation studies in the presence and absence of disease-associatedtarget mRNA were carried out. PKR protein was prepared as described inZheng and Bevilacqua (Zheng, X. and P. C. Bevilacqua. RNA 10:1934-1945(2004), herein incorporated by reference in its entirety). Protocols forPKR activation studies were based on Matsui, T. et al. (Matsui, T. etal. Biochem Biophys Res Comm 284:798-807 (2001), herein incorporated byreference in its entirety). Stock DNA/RNA solutions were prepared asfollows: hairpin 1 (“H1”), hairpin 2 (“H2”), disease-associated targetmRNA (“target mRNA fusion” comprising a target sequence with flankingregions), mRNA 1 (“healthy mRNA 1”), mRNA 2 (“health mRNA 2”), andhelper DNA (“D1” and “D2” annealed together in one solution). Acomparison antisense solution based on the protocol of Shir and Levitzki(Shir, A., and A. Levitzski. Nature Biotechnology 20:895-900 (2002),herein incorporated by reference in its entirety) was also prepared.Reactions were set up by combining the components as indicated below.The reactions were carried out using radioactive ATP solution andphosphatased PKR. Samples were then loaded onto prepared 10% SDS-PAGEgel and analyzed according to standard protein gel and radioactivevisualization protocols.

FIG. 7 illustrates the results of the PKR activation study for (a)Ewing's sarcoma and (b) glioma. The results show that RNA-HCR causes astrong activation of PKR in the presence of the target mRNA fusion (Lane3), but is not active in the absence of the target (Lane 2) or in thepresence of either healthy mRNA1 (Lane 4) or healthy mRNA 2 (Lane 5).The method of Shir and Levitzki is demonstrated in Lanes 6-9. PKRactivation in the presence of the target mRNA fusion (Lane 7) is not asstrong as using RNA-HCR (Lane 3). Also, the off states in the presenceof either healthy mRNA1 (Lane 8) or healthy mRNA2 (Lane 9) are not asgood as those observed using RNA-HCR (Lanes 4 and 5). As a result,RNA-HCR also provides a better on/off ratio for activation of PKR. Lanes10 and 11 show that PKR activation using RNA-HCR depends on the use ofboth hairpin species as expected.

Example 3 In Vivo HCR-RNA Activation of Protein Kinase PKR

HCR hairpins are delivered to target cells using standard gene deliverymethods. In the presence of an intracellular mRNA target, HCRpolymerization is initiated. The products act as PKR binding domains toactivate PKR. Activation of PKR leads to inhibition of translation andcell apoptosis in diseased cells containing the intracellular target.

Example 4 In Vivo RNA-HCR Therapy of Diseases Caused by Fused GeneMutations

Two or more genes are fused together to encode an oncogenic protein(Dohjima, T. et al. Molecular Therapy 7: 811-816 (2003), hereinincorporated by reference in its entirety). Although the two or moregenes are present in healthy cells, the overlap region between the genesis a distinct signature found only in tumor cells. HCR-RNA hairpinmonomers are designed to polymerize in the presence of a target sequenceincluded within the overlap region. The monomers have stems ranging inlength between 10 and 35 base pairs. The hairpin monomers are introducedin vivo into tumor cells by gene delivery methods known in the art.Recognition of the target overlap region by the HCR-RNA monomersinitiates hybridization chain reaction, which produces long nicked RNAduplexes in the tumor cells. This HCR product activates PKR in the tumorcells, which slows the production of all the proteins in the tumorcells. The activation of PKR also induces apoptosis. The triggering ofthis response slows and can reverse the growth of the tumor.

Example 5 HCR-Aptamer Therapy of Disease

An aptamer is identified that is able to specifically bind a targetmolecule within a diseased cell. HCR-RNA hairpin monomers are designedthat link the aptamer to a first RNA hairpin monomer in such a way thatin the absence of the target, the aptamer does not allow opening of thehairpin monomer and does not initiate HCR. The HCR-RNA hairpin monomersare introduced in vivo into cells by gene delivery methods known in theart. Recognition between the aptamer on a first RNA hairpin monomer andthe target allows opening of the first monomer, initiatingpolymerization by HCR and producing long nicked double-stranded RNA inthe target cells. The HCR product activates PKR in the target cells,which slows the production of all the proteins within the cells andinduces apoptosis. The triggering of this response slows and can reversethe growth of the diseased cells or kills the diseased cells.

1. A method of activating the RNA-dependent protein kinase PKR byhybridization chain reaction (HCR), the method comprising: contacting asample comprising PKR with a first metastable RNA hairpin monomercomprising an initiator complement region; and contacting the samplewith a second metastable RNA hairpin monomer, wherein the second monomercomprises a propagation region that is complementary to a portion of thefirst monomer; wherein a double-stranded RNA polymer comprising thefirst and second monomers is formed upon binding of an initiator to theinitiator complement region and the polymer activates PKR.
 2. The methodof claim 1, wherein the polymer comprises at least 35 base pairs.
 3. Themethod of claim 1, wherein the initiator is nucleic acid.
 4. The methodof claim 3, wherein the initiator is a portion of a target nucleic acid.5. The method of claim 4, further comprising: contacting the sample withat least one accessory molecule comprising a DNA sequence that binds toa region flanking the initiator.
 6. The method of claim 1, wherein theinitiator is able to bind to the initiator complement region when atarget is present in the sample.
 7. The method of claim 6, wherein thetarget is associated with a disease or disorder.
 8. The method of claim7, wherein the target is an mRNA associated with a cancer.
 9. The methodof claim 7, wherein the target is a viral nucleic acid.
 10. The methodof claim 1, wherein the initiator is part of a nucleic acid probe. 11.The method of claim 10, wherein upon binding of the nucleic acid probeto a target nucleic acid sequence the initiator is made available tobind to the initiator complement region of the first monomer.
 12. Themethod of claim 11, wherein the target is an mRNA molecule associatedwith a disease or disorder.
 13. The method of claim 6, wherein theinitiator comprises a recognition molecule such that upon binding of therecognition molecule to the target the initiator is made available tobind to the initiator complement region of the first monomer.
 14. Themethod of claim 13, wherein the recognition molecule is an aptamer. 15.The method of claim 13, wherein the target is a nucleic acid.
 16. Themethod of claim 13, wherein the target is selected from the groupconsisting of polypeptides, carbohydrates, lipids and small molecules.17. A method of treating a disease or disorder, comprising: providing afirst RNA hairpin monomer to a cell comprising a target nucleic acidassociated with the disease or disorder; and providing a second RNAhairpin monomer to the cell, wherein the second monomer comprises apropagation region that is substantially complementary to a portion ofthe first monomer; wherein the first and second monomers polymerize inthe presence of the target nucleic acid to form an RNA polymer thatactivates the RNA-dependent kinase PKR.
 18. The method of claim 17,wherein the disease or disorder is selected from the group consisting ofcancer and HIV infection.
 19. The method of claim 17, wherein the RNApolymer comprises at least 35 base pairs.
 20. The method of claim 17,wherein the method is carried out in vivo.
 21. The method of claim 17,wherein the method is carried out ex vivo.
 22. The method of claim 17,wherein the first RNA polymer comprises an initiator complement regionthat is complementary to a nucleic acid initiator.
 23. The method ofclaim 22, wherein the nucleic acid initiator is a portion of the targetnucleic acid sequence.
 24. The method of claim 23, further comprisingproviding the cell with least one accessory molecule comprising a DNAmolecule that binds to regions flanking the initiator.
 25. The method ofclaim 22, wherein the nucleic acid initiator is part of an initiationtrigger that is provided to the cell, wherein upon binding of the targetnucleic acid sequence to the initiation trigger, the initiator is madeavailable to initiate polymerization.
 26. The method of claim 25,wherein the initiation trigger comprises a recognition molecule.
 27. Themethod of claim 26, wherein the recognition molecule is an aptamer. 28.A kit for activating the RNA-dependent protein kinase PKR within asample comprising a target nucleic acid and PKR, the kit comprising: afirst RNA hairpin monomer comprising an initiator complement region; anda second RNA hairpin monomer comprising a propagation region that issubstantially complementary to a portion of the first monomer.
 29. Thekit of claim 28, wherein the initiator complement region iscomplementary to an initiator portion of the target nucleic acid. 30.The kit of claim 29, additionally comprising at least one accessorymolecule comprising a DNA molecule that is complementary to a regionflanking the initiator portion of the target nucleic acid.
 31. The kitof claim 28, wherein the target nucleic acid is associated with adisease or disorder
 32. The kit of claim 31, wherein the target nucleicacid is an mRNA associated with a cancer.
 33. The kit of claim 28,wherein the RNA hairpin monomers have a stem that is from 10 to 35 basepairs in length and a loop of at least 3 base pairs in length.
 34. Thekit of claim 28, further comprising a carrier that facilitatesintroduction of the composition into a cell.
 35. The kit of claim 34,wherein the carrier comprises one or more components selected from thefollowing: polymers, proteins, carbohydrates and lipids.
 36. The kit ofclaim 28, additionally comprising an initiation trigger, wherein aninitiator portion of the initiation trigger is complementary to theinitiator complement region of the first monomer.